Alzheimer's disease is the most common cause of dementia and affects roughly four million individuals. The ultimate goal of understanding the causes of Alzheimer's disease is to identify better methods of treatment and prevention. About 5 to 12% of Alzheimer's disease is due to autosomal dominant genes. The genes involved include the AD1 locus for the APP gene on chromosome 21 (St George-Hyslop et al. 1987) that codes for the Amyloid Precursor Protein (APP) (Karlinsky et al. 1992) and contains the sequences of amyloid-β (Aβ) present in senile plaques, the AD3 locus for PS-1 gene on chromosome 14 (Schellenberg et al. 1992; Alzheimer's Disease Collaborative Group 1995; Sherrington et al.1995; Sandbrink et al.1996) and the AD4 locus for PS-2 on chromosome 1 (Levy-Lahad et al.1995; Rogaev et al.1995).
The majority of Alzheimer's disease cases are sporadic. The AD2 locus for the APOE gene on chromosome 19 is an important locus for sporadic Alzheimer's disease (Saunders et al.1993; Saunders et al. 1996). The three major alleles are e2, e3 and e4, differing in two residues at position 112 and 158. The e4 allele is associated with an increased risk for Alzheimer's disease. It is present in 14% of normal Caucasians versus 37% in sporadic late-onset Alzheimer's disease (LOAD), and 48% in LOAD subjects with a family history of Alzheimer's disease (Saunders et al. 1993; Poirier et al.1993; Strittmatter et al. 1993). PS-1 has also been suggested as a risk factor for some cases of LOAD.
Based on studies of a number of different countries, factors such as dietary fat and total caloric intake have been shown to be highly correlated with the prevalence of Alzheimer's disease (Grant 1997). While this might seem to imply that environmental factors are more important than genetic ones, it is more likely that this represents the results of genetic environmental interaction. For example, a high fat diet may produce oxidative stress, and individuals genetically susceptible to such stress may be the ones who develop Alzheimer's disease. In the absence of a high fat diet the oxidative stress is too small to produce Alzheimer's disease even in genetically susceptible individuals.
Tau hyperphosphorylation. The presence of paired helical filaments (PHF) forming neurofibrillary tangles (NFTs) is one of the diagnostic characteristics of Alzheimer's disease (Iqbal et al.1986). NFTs are composed of an insoluble complex of microtubule (MT) proteins and hyperphosphorylated MT associated protein, tau. The degree of tau phosphorylation has been shown to correlate well with the degree of dementia (Iqbal et al.1986) and with the degree of minimal cognitive defect (MCI), a precursor to Alzheimer's disease (Arai et al.2000; Morris et al. 2001). Tau is moderately hyperphosphorylated in MCI and phosphorylated to an even greater degree in Alzheimer's disease (Su et al.1994; Ikura et al. 1998; Mailliot et al.1998; Spencer et al. 1964). Normally phosphorylated tau binds to tubulin and contributes to the assembly and stabilization of MT (Tseng et al. 1999). Hyperphosphorylated tau loses its ability to bind to tubulin, self-aggregates and contributes to the formation of PHF and NFTs (Iqbal et al.1986; Iqbal et al.1998).
APP phosphorylation. Okadaic acid (OA), is an inhibitor of protein phosphatases PP1 and PP2A. This compound has played an important role in the demonstration of the important role of phosphorylation of APP and other amyloid products in Alzheimer's disease. Holzer et al. (2000) examined the effect of OA on primary cultures of guinea pig neurons. Since guinea pig APP is 98% homologous to human APP at the protein level, identical regarding the Aβ sequence and is processed in a similar manner as human APP, this serves as a good animal model. Both intracellular and secreted APP was upregulated by OA treatment of 14 days old cultures in a concentration dependent manner.
Threonine(668) is within the carboxy-terminus of the Alzheimer's disease amyloid precursor protein (APP) and is a known in vivo phosphorylation site. Phosphorylation of APPthr(668) has been proposed to play a role in the regulation of APP function, metabolism and location (Iijima et al. 2000). APP is one of the rare proteins known to be phosphorylated within its ectodomain (Walter et al. 2000). Since Thr(688) is next to a proline, Standen et al. (2001) examined the potential role of proline-directed kinases in thr(688) phosphorylation. They found that stress-activated protein kinase-1b (SAPK1b) induced a robust phosphorylation of this site both in vitro and in vivo. They suggested that this finding provided a molecular framework to link cellular stresses with APP metabolism. Walter et al. (2000) have shown that membrane-bound beta APP as well as secreted forms of betaAPP can be phosphorylated by casein kinase (CK) 1- and CK2-like ectoprotein kinases.
Activation of the amyloid beta-protein precursor secretary pathway through alpha-secretase has been reported to increase the secretion of neuroprotective amyloid precursor protein and preclude the formation of amyloid beta-protein. Activation of protein kinase C has been shown to accelerate this secretory pathway (McLaughlin et al.1999). Thus, low levels of PKC would be expected to accelerate the deposition of Aβ. Kimura et al. (2000) showed that the levels of PKC were lower in Alzheimer's disease compared to control brains. Tan et al. (1997) has suggested that phosphorylation of Aβ plays a role in regulating its toxicity.
This is just a sampling of the many reports that suggest that in addition to the role of phosphorylation of tau in Alzheimer's disease, the phosphorylation of APP, Aβ and other amyloid related proteins also plays an important role in the etiology of Alzheimer's disease.
Okadaic acid and the genetics of Alzheimer's disease. Arendt et al.(1995) reported that chronic infusion into rat brain ventricles of OA resulted in a severe memory impairment, accompanied by a paired helical filament-like phosphorylation of tau protein and the formation of beta/A4-amyloid containing plaque-like structures in gray and white matter areas. In addition to its effect on the inhibition of phosphatases, OA directly or indirectly stimulates tau and other kinases. Adendt et al. suggested that an imbalance between protein phosphorylation [kinases] and dephosphorylation [phosphatases] might be crucial for the development of the molecular hallmarks of Alzheimer's disease.
Russ et al. (2001) recently examined the potential association between the glycogen synthase kinase 3β gene (GSK3B) and late onset Alzheimer's disease. They examined two SNPs in the promoter region, A/T-1727 and T/C-40. The frequency of the minor alleles were 0.13 and 0.35 respectively. They were in strong but not complete linkage disequilibrium (d′=0.48, p=≦10−7). Neither was significantly associated with late onset Alzheimer's disease (p=≦0.16 for both, odds ratios=1.3 and 1.2 respectively). They also identified three other rare SNPs with minor allele frequencies of less than 0.05. None of these were associated with Alzheimer's disease. This does not rule out a role of the GSK3B gene in Alzheimer's disease, since early Alzheimer's disease (EOAD) was not examined and combined the two common polymorphisms screened less than 60 percent of the possible haplotypes at this locus.
Tau is normally phosphorylated by the addition of phosphate groups to threonine and serine residues. The maintenance of normal levels of phosphorylation of tau is due to a balance between phosphorylation and dephosphorylation. Numerous candidate enzymes for the phosphorylation of tau have been proposed including glycogen synthase kinase 3 (Lovestone 1997), NCLK (CDK5 and p35/p25) (Hopkinson et al.1980; Sobue et al.2000; Kerokoski et al. 2001), CaMKII (Bennecib 2001), and others (Spencer et al. 1964; Tseng et al.1999; Lovestone1997; Guise S et al. 2001; Flaherty et al.2000). The kinases can be divided into proline-directed kinases and non-proline-directed kinases. Many of the serine and threonine residues are adjacent to prolines, implicating a role of proline-directed kinases. The proline directed kinases shown to play a role in tau phosphorylation include glycogen synthase kinase-3β (Lovestone 1997; Lovestone et al.1994; Sperber et al.1995), CDK5 and p35/p25 (Hopkinson et al.1980; Sobue et al.2000; Kerokoski et al. 2001; Pei et al.1998), CDC2 (Oawal et al.1992), p42 and p44 MAPK (Goedert et al.1992). The non-proline directed kinases include CaMKII (Bennecib et al. 2001), c-AMP-PK, PKC, casein kinases 1 and 2, TTK, PKN and p110MARK (Spencer et al.1964; Tseng et al.1999; Lovestone 1997; Guise et al.2001; Flaherty et al.2000; Taniguchi et al. 2001; Masliah et al.1990; Shapiro et al.1991).
Tau phosphatases. A number of enzymes that dephosphorylate tau have been identified (Lovestone 1997). These include the serine/threonine phosphatases PP1, PP2A, and PP2B (calcineurin). Sontag et al. (1996;1999) identified an important role of protein phosphatase 2A (PP2A) in the dephosphorylation of tau. The expression of PP2A is decreased in the hippocampus in Alzheimer's disease (Vogelsberg-Ragaglia et al. 2001). The catalytic unit of PP2A dephosphorylates tau serine 396 but not 199 and 202, while the holoenzyme dephosphorylates all three (Ono et al.1995). This was also the case for PP2B.
An additional candidate is acid phosphatase 1 (ACP1), a ubiquitous enzyme present in all tissues including the brain (Tanino et al.1999). ACP1 is also known as low molecular weight protein tyrosine phosphatase (LMWPTP). It shows no significant serine or threonine phosphatase activity (Zhang et al.1990; Chernoff et al. 1985) and thus is unlikely to be directly involved in tau phosphorylation. However, biochemical analysis and studies with specific antibodies to LMWPTP show that the level of ACP1 protein is significantly decreased in Alzheimer's disease brains (Shimohama et al. 1995; Shimohama et al.1993). ACP2, a lysosomal acid phosphatase, is associated with senile plaques in Alzheimer's disease (Omar et al.1993; Suzuki et al. 1967; Kawai et al.1992), and PTPRC, a protein tyrosine phosphatase receptor type also known as CD45, has also been implicated in Alzheimer's disease (Masliah et al. 1991).
The ACP1 gene spans 18 kb, 157 amino acids and consists of seven exons. Genetic variants of ACP1 have been recognized for many years. There are three major variants: ACP1*A, *B, and *C. Each of the three variants show two isoenzymes, slow and fast, due to a variant region spanning 34 nucleotides. This variant region is the result of alternate splicing such that two different exons, each 114 bp in length, are present in each isoform. ACP1*A differs from ACP1*B and *C by the presence of a Gln>Arg substitution at codon position 105 (Bryson et al.1995; Lazaruk et al.1993; Dissing et al.1992).
An A>G polymorphism of ACP1*A has been identified which creates a Taq I restriction endonuclease site in ACP1 that allows PCR based genotyping of ACP1. Restriction digestion of a 149 base pair(bp) PCR product from ACP1 with known oligonucleotide primers generated a 149 bp fragment from the intron 5′ of the C2 exon and into the C2 exon (Sensabaugh et al.1993). In the ACP1*A allele, the A>G sequence contains a target site for the restriction endonuclease Taq1. Cleavage of the 149 bp product with Taq1 enzyme generated 105 and 41 bp fragments when the ACP1*A allele was the substrate and ACP1*B and ACP1*C allele products were not cut with this enzyme(Id.).
Since ACP1*A has a lower enzyme activity than APC1*B or *C (Spencer et al.1964), there is a progressive decrease in ACP1 enzyme activity progressing from Taq I genotypes 11 (absence of *A variant) to 12 (50% *A variant) to 22 (100% *A variant). Thus, the term “2 allele” as used herein is meant to represent “ACP1*A” or “ACP1*A allele.”
Hyperphosphorylation of both tau and Aβ are proposed to be involved in the etiology of Alzheimer's disease. ACP1 is widely distributed in the brain and has been shown to be present in low levels in Alzheimer's disease brains. Polymorphisms of ACP1 are known to be associated with variations in enzyme activity, suggesting the possibility of an association of the ACP1*A allele of the ACP1 Taq I polymorphism with Alzheimer's disease.
Although NFT and senile plaques are characteristic of Alzheimer's disease, it has generally been thought that the Aβ cascade is most likely to be causative of Alzheimer's disease. It is consistent with many of the observations about the pathophysiology of Alzheimer's disease (Selkoe et al.2000). Despite this, a number of reports have shown that NFT rather than senile plaques more closely parallel both the duration and the severity of Alzheimer's disease (Arriagada et al.1992; Terry et al.1994; Braak et al.1996). One of the criticisms of a primary role of hyperphosphorylated tau in Alzheimer's disease has been the lack of evidence for a specific defective kinase or phosphatase in Alzheimer's disease (Daly et al. 2000).
Shirazi and Wood identified a subset of neurons in Alzheimer's disease brain that exhibited intense fyn tyrosine kinase immunoreactivity (Shirazi and Wood 1993). Double label immunohistochemistry showed that these fyn-positive neurons were also positive for hyperphosphorylated tau. They proposed that the activity of proline-directed tau kinases were regulated by fyn. APC1 may play an indirect role in the regulation of tau phosphorylation by counteracting the activity of fyn-kinase. Under this hypothesis low levels of ACP1 activity could play a role in the intense fyn immunostaining observed in some parts of Alzheimer's brain. Fyn is also able to directly phosphorylate tau at tyrosine residues. ACP1 could also be involved in direct dephosphorylation of such tau tyrosine residues. Studies of a synthetic portion of tau have shown that phosphorylation of a serine residue had little impact on conformation while phosphorylation of a tyrosine residue, resulted in considerable conformational change (Lee et al. 1998).
Some of the known substrates of ACP1 are the platelet derived growth factor (PDGF) and the insulin receptor (Taddei et al. 2000). ACP1 itself is phosphorylated, via Src and Src-related kinases, upon stimulation of cells with PDGF (Bucciantini et al. 1999; Cirri et al.1998). The observation of a significant association between the hypoactive variant of ACP1 is consistent with a direct or indirect role of this gene in the hyperphosphorylation of tau characteristic of NFTs or changes in the phosphorylation of APP playing a role in the production of Aβ.
While a single gene defect in a tau kinase or a tau phosphatase producing Alzheimer's disease has yet to be identified, the wide number of enzymes shown to phosphorylate and dephosphorylate tau and amyloid provide a rich substrate for an additive and epistatic effect of a series of genetic variants of the genes that regulate tau and APP phosphorylation. The present study suggests that an additive or epistatic effect of two or more genes, consisting of a combination of a hyperexpressed kinase and a hypoexpressed phosphatase could account for a sizeable proportion of the variance of Alzheimer's disease.